Skip to main content
SpringerLink
Log in

Detection of Fibroblast Growth Factor Receptor 1 (FGFR1) Transactivation by Muscarinic Acetylcholine Receptors (mAChRs) in Primary Neuronal Hippocampal Cultures Through Use of Biochemical and Morphological Approaches

  • Protocol
  • First Online:
Receptor-Receptor Interactions in the Central Nervous System

Part of the book series: Neuromethods ((NM,volume 140))

  • 648 Accesses

Abstract

In addition to their canonical intracellular signals involved in the regulation of neuronal plasticity, G-protein coupled receptors can also rapidly transactivate tyrosine kinase receptors and their downstream intracellular signaling in absence of specific ligands. Here we describe our protocol for dissociating and maintaining hippocampal primary neurons in high- and low-density culture, followed by a description of methods employed to evaluate neurite outgrowth and protein phosphorylation associated with fibroblast growth factor receptor 1 transactivation by muscarinic acetylcholine receptors. Our goal was to provide the reader with detailed protocols of the abovementioned techniques and to highlight advantages and limitations of the used approaches as compared to other valid alternatives.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Di Liberto V, Mudo G, Fuxe K, Belluardo N (2014) Interactions between cholinergic and fibroblast growth factor receptors in brain trophism and plasticity. Curr Protein Pept Sci 15(7):691–702

    Article  CAS  PubMed  Google Scholar 

  2. Tu H, Xu C, Zhang W, Liu Q, Rondard P, Pin JP, Liu J (2010) GABAB receptor activation protects neurons from apoptosis via IGF-1 receptor transactivation. J Neurosci 30(2):749–759. https://doi.org/10.1523/JNEUROSCI.2343-09.2010

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Borroto-Escuela DO, Romero-Fernandez W, Mudo G, Perez-Alea M, Ciruela F, Tarakanov AO, Narvaez M, Di Liberto V, Agnati LF, Belluardo N, Fuxe K (2012) Fibroblast growth factor receptor 1- 5-hydroxytryptamine 1A heteroreceptor complexes and their enhancement of hippocampal plasticity. Biol Psychiatry 71(1):84–91. https://doi.org/10.1016/j.biopsych.2011.09.012

    Article  PubMed  CAS  Google Scholar 

  4. Di Liberto V, Borroto-Escuela DO, Frinchi M, Verdi V, Fuxe K, Belluardo N, Mudo G (2017) Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures. Biochim Biophys Acta 1861(2):235–245. https://doi.org/10.1016/j.bbagen.2016.10.026

    Article  CAS  Google Scholar 

  5. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1(5):2406–2415. https://doi.org/10.1038/nprot.2006.356

    Article  PubMed  CAS  Google Scholar 

  6. Wallace TL, Johnson EM Jr (1989) Cytosine arabinoside kills postmitotic neurons: evidence that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis. J Neurosci 9(1):115–124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275

    PubMed  CAS  Google Scholar 

  8. Harada A, Teng J, Takei Y, Oguchi K, Hirokawa N (2002) MAP 2 is required for dendrite elongation, PKA anchoring in dendrites, and proper PKA signal transduction. J Cell Biol 158(3):541–549. https://doi.org/10.1083/jcb.200110134

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Pool M, Thiemann J, Bar-Or A, Fournier AE (2008) NeuriteTracer: a novel ImageJ plugin for automated quantification of neurite outgrowth. J Neurosci Methods 168(1):134–139. https://doi.org/10.1016/j.jneumeth.2007.08.029

    Article  PubMed  Google Scholar 

  10. Dotti CG, Sullivan CA, Banker GA (1988) The establishment of polarity by hippocampal neurons in culture. J Neurosci 8(4):1454–1468

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bartlett WP, Banker GA (1984) An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. II. Synaptic relationships. J Neurosci 4(8):1954–1965

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fletcher TL, De Camilli P, Banker G (1994) Synaptogenesis in hippocampal cultures: evidence indicating that axons and dendrites become competent to form synapses at different stages of neuronal development. J Neurosci 14(11 Pt 1):6695–6706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ziv NE, Smith SJ (1996) Evidence for a role of dendritic filopodia in synaptogenesis and spine formation. Neuron 17(1):91–102

    Article  CAS  PubMed  Google Scholar 

  14. Garner CC, Waites CL, Ziv NE (2006) Synapse development: still looking for the forest, still lost in the trees. Cell Tissue Res 326(2):249–262. https://doi.org/10.1007/s00441-006-0278-1

    Article  PubMed  Google Scholar 

  15. Banker G, Goslin K (1988) Developments in neuronal cell culture. Nature 336(6195):185–186. https://doi.org/10.1038/336185a0

    Article  PubMed  CAS  Google Scholar 

  16. Benson DL, Watkins FH, Steward O, Banker G (1994) Characterization of GABAergic neurons in hippocampal cell cultures. J Neurocytol 23(5):279–295

    Article  CAS  PubMed  Google Scholar 

  17. Biffi E, Regalia G, Menegon A, Ferrigno G, Pedrocchi A (2013) The influence of neuronal density and maturation on network activity of hippocampal cell cultures: a methodological study. PLoS One 8(12):e83899. https://doi.org/10.1371/journal.pone.0083899

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Banker GA, Cowan WM (1977) Rat hippocampal neurons in dispersed cell culture. Brain Res 126(3):397–342

    Article  CAS  PubMed  Google Scholar 

  19. Yang Q, Ke Y, Luo J, Tang Y (2017) Protocol for culturing low density pure rat hippocampal neurons supported by mature mixed neuron cultures. J Neurosci Methods 277:38–45. https://doi.org/10.1016/j.jneumeth.2016.12.002

    Article  PubMed  CAS  Google Scholar 

  20. Banker GA (1980) Trophic interactions between astroglial cells and hippocampal neurons in culture. Science 209(4458):809–810

    Article  CAS  PubMed  Google Scholar 

  21. Ray J, Peterson DA, Schinstine M, Gage FH (1993) Proliferation, differentiation, and long-term culture of primary hippocampal neurons. Proc Natl Acad Sci U S A 90(8):3602–3606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Grabrucker A, Vaida B, Bockmann J, Boeckers TM (2009) Synaptogenesis of hippocampal neurons in primary cell culture. Cell Tissue Res 338(3):333–341. https://doi.org/10.1007/s00441-009-0881-z

    Article  PubMed  Google Scholar 

  23. Brewer GJ, Torricelli JR, Evege EK, Price PJ (1993) Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 35(5):567–576. https://doi.org/10.1002/jnr.490350513

    Article  PubMed  CAS  Google Scholar 

  24. Dotti CG, Banker GA (1987) Experimentally induced alteration in the polarity of developing neurons. Nature 330(6145):254–256. https://doi.org/10.1038/330254a0

    Article  PubMed  CAS  Google Scholar 

  25. Goslin K, Banker G (1989) Experimental observations on the development of polarity by hippocampal neurons in culture. J Cell Biol 108(4):1507–1516

    Article  CAS  PubMed  Google Scholar 

  26. Bradke F, Dotti CG (1997) Neuronal polarity: vectorial cytoplasmic flow precedes axon formation. Neuron 19(6):1175–1186

    Article  CAS  PubMed  Google Scholar 

  27. Basarsky TA, Parpura V, Haydon PG (1994) Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution. J Neurosci 14(11 Pt 1):6402–6411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141(7):1117–1134. https://doi.org/10.1016/j.cell.2010.06.011

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Ubersax JA, Ferrell JE Jr (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8(7):530–541. https://doi.org/10.1038/nrm2203

    Article  PubMed  CAS  Google Scholar 

  30. Iliuk A, Martinez JS, Hall MC, Tao WA (2011) Phosphorylation assay based on multifunctionalized soluble nanopolymer. Anal Chem 83(7):2767–2774. https://doi.org/10.1021/ac2000708

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. de Graauw M, Hensbergen P, van de Water B (2006) Phospho-proteomic analysis of cellular signaling. Electrophoresis 27(13):2676–2686. https://doi.org/10.1002/elps.200600018

    Article  PubMed  CAS  Google Scholar 

  32. Iliuk A, Galan J, Tao WA (2009) Playing tag with quantitative proteomics. Anal Bioanal Chem 393(2):503–513. https://doi.org/10.1007/s00216-008-2386-0

    Article  PubMed  CAS  Google Scholar 

  33. Kruger R, Zinn N, Lehmann WD (2009) Quantification of protein phosphorylation by microLC-ICP-MS. Methods Mol Biol 527:201–218., ix. https://doi.org/10.1007/978-1-60327-834-8_15

  34. Czernik AJ, Girault JA, Nairn AC, Chen J, Snyder G, Kebabian J, Greengard P (1991) Production of phosphorylation state-specific antibodies. Methods Enzymol 201:264–283

    Article  CAS  PubMed  Google Scholar 

  35. Mohammadi M, Dikic I, Sorokin A, Burgess WH, Jaye M, Schlessinger J (1996) Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. Mol Cell Biol 16(3):977–989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Belluardo N, Wu G, Mudo G, Hansson AC, Pettersson R, Fuxe K (1997) Comparative localization of fibroblast growth factor receptor-1, -2, and -3 mRNAs in the rat brain: in situ hybridization analysis. J Comp Neurol 379(2):226–246

    Article  CAS  PubMed  Google Scholar 

  37. Ignatoski KM (2001) Immunoprecipitation and western blotting of phosphotyrosine-containing proteins. Methods Mol Biol 124:39–48

    PubMed  CAS  Google Scholar 

  38. Hopkins AM, DeSimone E, Chwalek K, Kaplan DL (2015) 3D in vitro modeling of the central nervous system. Prog Neurobiol 125:1–25. https://doi.org/10.1016/j.pneurobio.2014.11.003

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy

    Valentina Di Liberto, Giuseppa Mudó & Natale Belluardo

  2. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    Dasiel O. Borroto-Escuela

  3. Department of Biomolecular Science, Section of Physiology, University of Urbino, Urbino, Italy

    Dasiel O. Borroto-Escuela

  4. Observatorio Cubano de Neurociencias, Grupo Bohío-Estudio, Yaguajay, Cuba

    Dasiel O. Borroto-Escuela

  5. Department of Neuroscience, Karolinska Institutet, Solna, Sweden

    Kjell Fuxe

Authors
  1. Valentina Di Liberto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Giuseppa Mudó
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dasiel O. Borroto-Escuela
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kjell Fuxe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Natale Belluardo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Natale Belluardo .

Editor information

Editors and Affiliations

  1. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    KJELL FUXE

  2. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    Dasiel O. Borroto-Escuela

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Di Liberto, V., Mudó, G., Borroto-Escuela, D.O., Fuxe, K., Belluardo, N. (2018). Detection of Fibroblast Growth Factor Receptor 1 (FGFR1) Transactivation by Muscarinic Acetylcholine Receptors (mAChRs) in Primary Neuronal Hippocampal Cultures Through Use of Biochemical and Morphological Approaches. In: FUXE, K., Borroto-Escuela, D. (eds) Receptor-Receptor Interactions in the Central Nervous System. Neuromethods, vol 140. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8576-0_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8576-0_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8575-3

  • Online ISBN: 978-1-4939-8576-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation